Light Microscopy Flashcards
What size can the eye see down to
0.2mm
Light microscopy can see down to
0.2um
Electron microscope can see down to
0.2nm
Magnification
Ratio of size of image to object
Total = power of objective lens x power of eyepiece
Resolution
Clarity of image, illumination and quality of optics
Resolution (D) =
(0.61 x wavelength)/NA
NA = N x sina
NA is numerical aperture and labelled on the objective lens
a is angular apeture and N is the refractive index of the medium between the specimen and objective lens
Smaller D = better resolution
Contrast
contrast between lightest and darkest area of sample
Numerical aperture
size of cone of light coming from each point of the specimen
Hooke
First named a cell
Simple microscope with oil lamp as a light source
Leeuwenhoek
improved production of lenses and at over 200 x magnification observed cell types
Zeiss
Made lenses allowing resolution at theoretical limits
Components of light microscopy
Objective lens,( collects cone of light rays to make an image), condenser lens (focuses cone of light rays on specimen), eyepiece and light source
Brightfield light microscopy
Poor images of living cells compared to phase contrast and DIC
Phase contrast
High contrast images
density of specimen determines diffraction of light
Diffracted and undiffracted rays give rise to changes in brightness- contrast
Degree of darkness/brightness depends on refractive index of a region
Refracted and unrefracted light recombine at image plane to form the image
DIC
use polarised light - polariser
Prism splits source of light into 2 parts, 2nd prism recombines
If one of beams is diffracted by the specimen, when they are recombined they will interfere and generate contrast ranging from black to white
If beams not diffracted they don’t interfere when recombined and produce pale grey
Contrast generated by difference in refractive index of specimen and surrounding medium
Tissue sectioning
cut into thin slices
Detection of cellular components/ viability with bright field using:
dyes
chromogenic (production of colour or pigments) enzyme substrate
Dyes
Hysto or cyto chemical dyes
Give rise to rust colour
Interact with phenolic compounds in cells and lignin in CWs
Which types of microscopy can be used in time lapse microscopy
Phase contrast and DIC - produces movie through multiple images
Fluoresence
Absorbing light of a particular wavelength then emitting light of a different colour and wavelength
fluoresence vs bright field
Main difference is use of selective filters and directing excitation light through objective lens, into the sample and observing emitted fluorescent light passing back through objective lens from sample.
Excitation light reflected onto dichroic mirror
Applications of fluorescence microscopy
visualising organelles
Different dyes for relative localisation of molecules
Autofluorescence can reveal certain cell components
Immunofluorescence microscopy used to detect specific proteins with an antibody to which a fluorescent dye has been covalently attached
Confocal microscopy
limitation of fluorescence: fluorescent light come not only from plane of focus but also molecules above and below- see blurred image due to fluorescent images from many depths
Better resolution because only plane in focus is visible
More advanced, images in multiple planes
Image live specimens, take images at multiple intervals
Uses of confocal
used in co-localization, intracellular, thick specimens
Co-localization
observation of the spatial overlap between two (or more) different fluorescent labels, each having a separate emission wavelength, to see if the different “targets” are located in the same area of the cell or very near to one another.
